双抗夹心ELISA检测食品中大肠杆菌O157:H7方法研究

研究获得纯化抗大肠杆菌O157:H7IgY抗体,经检测10mg/ml纯化IgY抗体的效价为1:320;以大肠杆菌O157:H7免疫新西兰大耳白兔,获得兔抗大肠杆菌O157:H7IgG抗体,效价达1:25600。以兔抗大肠杆菌O157:H7IgG抗体稀释3200倍作为捕获抗体,抗大肠杆菌O157:H7IgY抗体为检测抗体建立双抗夹心ELISA方法检测大肠杆菌O157:H7,正交试验分析表明,捕获抗体于37℃包被2h、不封闭、抗原与捕获抗体于37℃结合2h、检测抗体浓度为0.25mg/ml、与抗原于37℃结合1h为最优反应条件。该方法对纯培养菌液检出限为105CFU/ml,具有良好的敏感性及特异性。染菌样品经在EC增菌液中选择性培养后进行双抗夹心ELISA检测,接种量为0.1～1CFU/g(ml)的样品在培养12h后可检出阳性反应,1～10CFU/g(ml)的样品在培养8h后可检出阳性反应。     

发酵大豆食品中染料木素含量的ELISA测定

染料木素是大豆发酵食品中的主要异黄酮成分。制备了牛血清白蛋白-染料木素免疫抗原和卵白蛋白-染料木素包被抗原,并对它们进行紫外和SDS-聚丙稀酰胺凝胶电泳鉴定。抗染料木素抗体的效价为1:1600。建立竞争ELISA测定发酵大豆制品中染料木素含量方法,染料木素测定浓度范围为6.25～150μg/L;回收率为87%～102%;亲和常数为1.62×108;与大豆甙元、芒柄花素的交叉反应率分别为9.8%、4.57%;与槲皮素、儿茶素、单宁和核黄素等结构类似物无交叉反应。同时将ELISA法与薄层层析法进行比较。     

Mechanism of action of Melaleuca armillaris (Sol. Ex Gaertu) Sm. essential oil on six LAB strains as assessed by multiparametric flow cytometry and automated microtiter-based assay

Little is known concerning the effects of essential oils (EOs) against lactic acid bacteria (LAB), either of the human gastrointestinal microflora or those involved in industrial processes. GC and GC/MS analysis of Melaleuca armillaris EO resulted in the identification of 68 compounds comprising 99.6% of the oil. Eucalyptol (1,8-cineole) was the major compound (68.9%) and the composition was largely dominated by the oxygenated monoterpenes fraction (77.3%). The anti-LAB activities of the EO were first checked by the disc diffusion assay. In a second phase, time-survival kinetics of each strain incubated with increasing concentrations of the EO (0.25, 2.5, 5 and 25 μg/ml) were established using an automated microtiter assay (Bioscreen C). Bacteriostatic or bactericidal effects were noticed depending on the studied strain and on the applied concentration of the EO. The mathematical modelling of the kinetics showed that in presence of increasing concentrations of M. armillaris EO, the lag phases of growth were extended (0.69%–97.5%) and both the growth rate and final cell density were reduced. Variations depending on the strain were noticed.     

Antioxidant activities of aqueous extracts of selected plants

The antioxidant properties of 25 edible tropical plants, expressed as Trolox equivalent antioxidant capacity (TEAC), were studied using DPPH (1,1-diphenyl-2-picrylhydrazyl free radical) scavenging and reducing ferric ion antioxidant potential (FRAP) assays. Their cupric ion chelating activities (CCA) and total polyphenol contents (TPC) were also determined. A strong correlation between TEAC values obtained for the DPPH assay (TEAC_DPPH) and those for the FRAP assay (TEAC_FRAP) implied that compounds in the extracts were capable of scavenging the DPPH free radical and reducing ferric ions. A satisfactory correlation of TPC with TEAC_DPPH and TEAC_FRAP suggested that polyphenols in the extracts were partly responsible for the antioxidant activities while its correlation with CCA was poor, indicating that polyphenols might not be the main cupric ion chelators. Principal component analysis (PCA) indicated that TEAC_DPPH, TEAC_FRAP and TPC contributed to the total variation in the antioxidant activities of the plants.     

保健品中牛羊源性成分的PCR检测

本文根据牛、羊线粒体mtDNA中的保守序列，设计了针对牛羊源性成分检测的特异性扩增引物，通过聚合酶链反应技术(Polymerase Chain Reaction，PCR)建立了保健品中牛羊源性成分的快速检测方法。通过内切酶DpnⅡ和SspⅠ可分别对牛羊成分的扩增结果进行进一步验证，该方法对牛源性成分的检测低限为0．05％，对山羊和绵羊源性成分的检测低限分别为0．005％和和0．5％，可作为保健品中牛羊源性成分检测有效方法，也可作为保健品及动物源性产品中成分真伪鉴别的准确、可靠方法。     

除草剂莎稗磷多克隆抗体的制备

根据莎稗磷的化学结构特征,设计合成莎稗磷半抗原,采用活化酯法与牛血清白蛋白偶联,免疫新西兰大耳白兔获得抗莎稗磷多克隆抗血清用于免疫分析试验。抗血清效价高达1∶160 000,100μg/L莎稗磷的抑制率达到了59.76%。抗体经Protein A-Sepharose 4B亲和层析柱纯化后,50μg/L莎稗磷的抑制率提高到92%,其它有机磷农药及结构类似化合物与抗体的交叉反应率均小于0.01%,表明了所制备莎稗磷抗体的高特异性。     

采用GC-MS和ELISA试剂盒法对比分析白酒中的塑化剂

使用GC-MS和ELISA试剂盒法对白酒中的塑化剂进行测定,考察了GC-MS的精确度及回收率,ELISA试剂盒法的标准曲线,并比较了两种方法的不同前处理对测定结果的影响。结果表明:GC-MS的精密度标准偏差在-6.0%~6.3%,回收率在87%~113%,ELISA试剂盒法线性良好,两种方法对样品的测定结果接近,表明两种方法都可以对白酒中的塑化剂进行定性定量分析。ELISA试剂盒法前处理比GC-MS简便,但有一定的损失,可以进行快速的定性定量分析,如需更准确的测定塑化剂含量,可以配合GC-MS法进行验证检测。     

Temperature gradients drive radial fluid flow in petri dishes and multiwell plates

Liquid in a Petri dish spontaneously circulates in a radial pattern, even when the dish is at rest. These fluid flows have been observed and utilized for biological research, but their origins have not been well‐studied. Here, particle‐tracking to measure velocities of radial fluid flows, which are shown to be linked to evaporation, is used. Infrared thermal imaging was used to identify thermal gradients at the air‐liquid interface and at the bottom of the dish. Two‐color ratiometric fluorescence confocal imaging was used to measure thermal gradients in the vertical direction within the fluid. A finite‐element model of the fluid, incorporating the measured temperature profiles, shows that buoyancy forces are sufficient to produce flows consistent with the measured particle velocity results. Such flows may arise in other dish or plate formats, and may impact biological research in positive or negative ways. © 2016 American Institute of Chemical Engineers AIChE J, 62: 2227–2233, 2016     

A Comparison of Fresh Frozen vs. Formalin-Fixed,Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer.